Elt University and by EFRO through the Interreg V Grensregio Vlaanderen Nederland project Trans Tech Diagnostics.LBP.Free flow electrophoresis permits preparation of EV fractions with high recovery and purity rates Gerhard Weber1, Robert Wildgruber1, Simon Staubach2, Robin Dittrich3, Peter Horn3, Verena Boerger4 and Bernd Giebel2 FFE Service; 2Institute for Transfusion Medicine, University Hospital Essen, University of Ubiquitin-Specific Peptidase 18 Proteins MedChemExpress Duisburg-Essen, Essen, Germany; Division of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; 3Institute for Transfusion Medicine, University Hospital Essen, University of DuisburgEssen, Essen, German; 4Institute for Transfusion Medicine, University Hospital Essen, University Duisburg-Essen, Essen, GermanyIntroduction: Presently, it remains a challenge to prepare extracellular vesicles (EVs), specifically these from physique fluids, like plasma, to higher purity. Neither fractionation by density nor by size is sufficient to separate EVs from all contaminants e.g. high and low density lipoprotein (HDL/LDL) along with other contaminating components. For now, a timeconsuming mixture of two approaches (density and size) is required to enrich EVs to higher purities, frequently resulting in low EV recoveries. Totally free Flow Electrophoresis is often a well-established preparative and micropreparative technique to separate analytes with inherent distinction of charge density and/or difference of pI-value. Procedures: Absolutely free Flow Interval Zone Electrophoresis (FF-IZE), using media of diverse pH-values, ranging from pH = 8 to pH = four.eight offers most suitable protocols for the quantitative separation of amphoteric analytes,Thursday May possibly 18,like proteins and peptides from non-amphoteric analytes like lipid vesicles, DNA and RNA. Results: Inside our ongoing project we have optimized FF-IZE-pH protocols for the purification and isolation of EVs also DNA and RNA from cell culture supernatants and human plasma samples. Upon screening for EV-specific samples inside a dot blot technique, EV-specific antigens are especially recovered inside a chosen number of fractions. Currently, we characterize the identified fractions in more detail. For the enumeration of ready EVs we make use of the Nanoparticle Tracking Evaluation (NTA). Additionally, the presence of EV markers plus the absence of contaminants are analyzed by Western Blot. We document the appearance of TR alpha 1 Proteins custom synthesis isolated EVs by transmission electron microscopy and decide the miRNA profiles on the obtained fractions. Summary/Conclusion: The principle of FFE, the EV isolation strategy and our ongoing outcomes will likely be presented.Funding Supported by the Polish National Centre for Analysis and Improvement STRATEGMED1/235773/19/NCBR/2016 “EXPLORE ME”.LBP.MicroRNA biogenesis and heterogeneous miRNA distribution in cancer EVs Nils J. Groenewegen, Catrin Lutz, Alba M. Losada, Monique A.J. van Eijndhoven and D. Michiel Pegtel Exosomes Investigation Group, Division of Pathology, VU University Medical Center, Amsterdam, The NetherlandsLBP.Visualization of extracellular vesicles derived from human bone marrow mesenchymal stem cells making use of fluorescent and magnetic labels; in vitro and in vivo studies Sylwia Koniusz1, Anna Andrzejewska1, Andrea Del Fattore2, Elbieta Karnas3, Malgorzata Frontczak-Baniewicz4, Hanna Kozlowska5, Maurizio Muraca6, Miroslaw Janowski7 and Barbara Lukomska1 NeuroRepair Department, Mossakowski Health-related Analysis Centre, PAS, Warsaw, Poland; 2Multifactorial Disease and Complicated Phenotype Investigation Area, Bambino GesChildren’.